Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:An analogue test material was considered to be non-mutagenic under the conditions of this test (OECD 471, EU Method B13/14, OCSPP 870.5100 and relevant Japanese guidelines).

 

Chromosome aberration test:An analoguetest item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro (OECD 473 and relevant Japanese guidelines).

 

Mouse lymphoma assay:An analogue test item did not induce any toxicologically significant increases in the mutant frequency at

the TK +/- locus in L5178Y cells (OECD 476, EU Method B.17 and OPPTS 870.5300).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2017 to 10 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / β-naphtha flavone induced S9 mix
Test concentrations with justification for top dose:
- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 2: 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
Dimethyl formamide
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl formamide
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG (CAS number 4245-77-6; Batch number 67F-3700; Purity treated as 100 %; Solvent DMSO)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
9AA (CAS number 90-45-9; Batch S32398-438; Purity 99.9 %; Expiry date 01 October 2017; Solvent DMSO)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4NQO (CAS number 56-57-5; Batch 030M1206; Purity 100 %; Expiry date 08 October 2017; Solvent DMSO)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
2AA (CAS number 613-13-8; Batch STBB1901M9; Purity 97.5 %; Expiry date 08 October 2017; Solvent DMSO)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
BP (CAS number 50-32-8; Batch 090M1400V; Purity 96 %; Expiry date 12 October 2017; Solvent DMSO)
Details on test system and experimental conditions:
STUDY CONTROLS
- The solvent (vehicle) control used was dimethyl formamide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
- The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

STERILITY CONTROLS
- Top agar and histidine/biotin or tryptophan in the absence of S9-mix (in triplicate).
- Top agar and histidine/biotin or tryptophan in the presence of S9-mix (in triplicate).
- The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions were pre-prepared using standardised in-house procedures (outside the confines of this study).
- Lot Numbers 31 March 2017 (Experiment 1) and 21 October 2016 (Experiment 2) were used in this study.
- Copies of the S9 Certificates of Efficacy are presented in Appendix 2 (attached).

S9 MIX AND AGAR
- The S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of the test.
- The S9 mix contained S9 (5.0 mL); 1.65 M KCl/0.4 M MgCl2 (1.0 mL); 0.1 M glucose-6-phosphate (2.5 mL); 0.1 M NADP (2.0 mL); 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL); sterile distilled water (14.5 mL).
- A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- Top agar was prepared using 0.6 % Bacto agar (lot number 6147883 03/21) and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (Lot numbers 44593 07/17 and 44674 07/17).

BACTERIA
- The five strains of bacteria used are shown in the table below together with their mutations.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain L T2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAI 00~ the R-factor plasmid pKMIOI enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains auvrA-DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; Lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

TEST ITEM PREPARATION AND ANALYSIS
- In solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in dimethyl formamide at 50 mg/mL. Dimethyl formamide was, therefore, selected as the vehicle. The test item was originally noted as miscible in acetone at 100 mg/mL but the formulation came out of solution and would not reform as a solution.
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction was required for test item purity. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
- All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This was an exception with regard to GLP and was been reflected in the GLP compliance statement.

DOSE SELECTION FOR EXPERIMENT 1
- Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

EXPERIMENT 1 - WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL) of the appropriate concentration of test item or solvent vehicle or 0.1 mL of appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

EXPERIMENT 1 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that following addition of the test item formulation and bacterial culture, S9-mix (0.5 mL) was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

EXPERIMENT 1 - INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).
- Manual counts were performed at 5000 μg/plate because of a test item film.

DOSE SELECTION FOR EXPERIMENT 2
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
- Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

EXPERIMENT 2 - WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).
- Manual counts were performed at 5000 μg/plate because of a test item film. Several additional manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

ACCEPTANCE CRITERIA
- The reverse mutation assay may be considered valid if the following criteria are met:
(i) All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
(ii) All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are TA 1535 (7 to 40); TA100 (60 to 200); TA1537 (2 to 30); TA98 (8 to 60); WP2uvrA (10 to 60). Combined historical negative and solvent control ranges for 2012 and 2013 are presented in Appendix 1 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E09 bacteria per mL.
(iv) Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2015 and 2016 are presented in Appendix 1 (attached).
(v) There should be a minimum of four non-toxic test item dose levels.
(vi) There should be no evidence of excessive contamination.

MAJOR COMPUTERISED SYSTEMS
- Ames Study Manager and Sorcerer Imaging System.
- Delta Building Monitoring System.
Evaluation criteria:
EVALUATION CRITERIA
- There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
(i) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(ii) A reproducible increase at one or more concentrations.
(iii) Biological relevance against in-house historical control ranges.
(iv) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(v) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by · an out-of-historical range response (Cariello and Piegorsch, 1996).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
STATISTICAL ANALYSIS
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- The vehicle (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 (attached) for Experiment 1 and Table 4 and Table 5 (attached) for Experiment 2.
- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1 (attached).
- The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film (creamy in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.
- There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.
Conclusions:
The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

GUIDELINE

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

 

RESULTS

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film (creamy in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

 

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

 

CONCLUSION

The test item was considered to be non-mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 April 2015 to 23 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: chromosome aberration
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 °C with 5% C02 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/f3-Naphthoflavone induced rat liver homogenate metabolising system (S9),
Test concentrations with justification for top dose:
MAIN TEST
- 4(20)-hour without S9: 0,5, 10,20,40,80, 160 μg/mL
- 4(20)-hour with S9 (2%): 0,5, 10,20,40,80, 160 μg/mL
- 24-hour without S9: 0,5, 10,20,40,80, 160 μg/mL
-
Vehicle / solvent:
Acetone
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Remarks:
Fisher Scientific (Batch 1408955)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
mitomycin C
Remarks:
MMC (Sigma; Batch SLBH9906V; Solvent Minimal Essential Medium)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
CP (Sigma; Batch MKBS0021V; Solvent dimethyl sulphoxide)
Details on test system and experimental conditions:
CELLS
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
- The donor for the preliminary test was male (aged 29 years) and the donor for the main experiment was male (aged 27 years).

MICROSOMAL ENZYME FRACTION
- Lot No PB/f3NF S9 23/11/14 S9 was used in this study.
- The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/f3-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCl solution (lg liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
- The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20 % (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (5 mM). The final concentration of S9, when dosed at a 10 % volume of S9-mix into culture media, was 2 %.
- This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.

TEST ITEM PREPARATION AND ANALYSIS
- The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 μg/mL. However, due to the use of acetone as the solvent, which can be used at a maximum dose concentration of 0.5 %, the maximum achievable dose level was 2500 μg/mL.
- Prior to each experiment, the test item was accurately weighed, formulated in acetone and
appropriate serial dilutions prepared.
- The solubility of the test item was investigated in a separate Mouse Lymphoma Assay conducted by Harlan Laboratories Ltd.
- There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
- The pH and osmolality readings are presented in the table below.
- The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

CULTURE CONDITIONS
- Duplicate lymphocyte cultures (A and B) were established for each dose level.
- Each culture dispensed into plastic flasks contained MEM 10 % FBS (9.05 mL); Li-heparin (0.1 mL); phytohaemagglutinin (0.1 mL); heparinised whole blood (0.75 mL).

EXPOSURE WITH METABOLIC ACTIVATION (S9)
- After approximately 48 hours incubation at approximately 37 °C, 5 % C02 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20 % S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
- After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5 % C02 in humidified air.

EXPOSURE WITHOUT METABOLIC ACTIVATION (S9)
- For the 4(20)-hour exposure in the absence of 89 - After approximately 48 hours incubation at approximately 37 °C with 5 % C02 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
- After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
- For the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.95 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 5 % C02 in humidified air for 24 hours.
- The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

PRELIMINARY TOXICITY TEST
- Three exposure groups were used:
(i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
(ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
(iii) 24-hour continuous exposure to the test item without S9-mix.
- The dose range of test item used was 9.77 to 2500 μg/mL.
- Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
- Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

MAIN EXPERIEMENT
- Three exposure groups were used for the Main Experiment:
(i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 5, 10, 20, 40, 80 and 160 μg/mL.
(ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 5, 10, 20, 40, 80 and 160 μg/mL.
(iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 5, 10, 20, 40, 80 and 160 μg/mL.
- Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two and a half hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. - After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative.
- Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

STAINING
- When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

QUALITATIVE SLIDE ASSESSMENT
- The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

CODING
- The slides were coded using a computerised random number generator.
- Supplementary slides were coded manually.

MITOTIC INDEX
- A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING OF CHROMOSOME DAMAGE
- Where possible the first 150 consecutive well-spread metaphases from each culture were counted, where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1).
- Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including endoreduplicated cells) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 2 (attached).

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
- Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.
- A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The increase observed is considered to be dose-related.
- When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
- Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploid and endoreduplications.
Evaluation criteria:
EVALUATION CRITERIA
- The following criteria were used to determine a valid assay:
(i) The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
(ii) All the positive control chemicals induced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
(iii) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
(iv) The required number of cells and concentrations were analysed.
Statistics:
STATISTICAL ANALYSIS
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
- A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
- The dose range for the Preliminary Toxicity Test was 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL. The maximum dose was the maximum achievable dose level, due to formulation difficulties and the necessity of using acetone as the solvent.
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the
exposure, at and above 156.25 μg/mL and 78.13 μg/mL in the 4(20)-hour exposure groups in the
absence and presence of metabolic activation, respectively and at and above 39.06 μg/mL in the
continuous exposure group.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 μg/mL in all three exposure groups. The mitotic index data are presented in Table 1 (attached). The test item induced some evidence of toxicity in the 24-hour exposure group only.
- The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 160 μg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.
 
CHROMOSOME ABERRATION TEST – MAIN EXPERIMENT
- The dose levels of the controls and the test item are given in the table below.
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level of test item, 160 μg/mL in all three exposure groups.
- Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 80 μg/mL, in the 4(20)-hour exposure group in the absence of S9, and at 160 μg/mL, in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group.
- The mitotic index data for the Main Experiment are given in Table 2 and Table 3 (attached). They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in the 4(20)-hour exposure groups in the absence or presence of S9. Some toxicity was demonstrated throughout the dose range of the 24-hour continuous exposure group and a 42 % reduction in mitotic index was achieved at 160 μg/mL.
- The maximum dose level selected for metaphase analysis was the lowest precipitating dose level and was 80 μg/mL for the 4(20)-hour exposure group in the absence of S9, and was 160 μg/mL, for the 4(20)-hour exposure group in the presence of S9 and for the 24-hour continuous exposure group.
- The chromosome aberration data are given in Table 4, Table 5 and Table 6 (attached). The assay was considered valid as it met all of the following criteria:
(i) The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
(ii) All the positive control chemicals induced a demonstrable positive response (p ≤ 0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
(iii) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
(iv) The required number of cells and concentrations were analysed.
- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The polyploid cell frequency data are given in Table 7 (attached). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the three exposure groups.

DOSE LEVELS OF CONTROLS AND TEST ITEM

Group

Final concentration of test item (µg/mL)

4(20)-hour without S9

0*, 5, 10, 20*, 40*, 80*, 160, MMC 0.4*

4(20)-hour with S9 (2 %)

0*, 5, 10, 20, 40*, 80*, 160*, CP 2*

24-hour without S9

0*, 5, 10, 20*, 40*, 80*, 160*, MMC 0.2*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

GUIDELINE

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 26 September 2014 and Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.

 

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, the main experiment was performed using three exposure conditions; 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration with cell harvest after a 20-hour expression period, a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels selected for the main experiment were based on the results of the preliminary toxicity test and were limited to include the lowest precipitating dose level. The dose levels selected for the main experiment were 0,5, 10, 20, 40, 80, 160 μg/mL for the 4(20)-hour without S9, 4(20)-hour with S9 (2%) and 24-hour without S9 exposure groups.

 

RESULTS

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did demonstrate some toxicity in the preliminary toxicity test in the 24-hour exposure group only, however, the dose range for the main experiment was limited to include the lowest precipitating dose level, irrespective of toxicity, as directed in the OECD 473 guideline. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

 

CONCLUSION

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13
Reason / purpose:
read-across source
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March 2015 to 26 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mouse lymphoma assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
3.7.2c
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / ß-Naphtha flavone induced S9 mix
Test concentrations with justification for top dose:
- Experiment 1 (4-hour without S9): 78.13, 156.25, 312.5, 468.74, 546.87, 625 μg/mL
- Experiment 1 (4-hour with S9 2%): 19.53, 39.06, 78.13, 156.25, 312.5, 468.74 μg/mL
- Experiment 2 (24-hour without S9): 39.06, 78.13, 156.25, 312.5, 390.61 μg/mL
- Experiment 2 (4-hour with S9 2%): 78.13, 156.25, 312.5, 390.61, 468.74, 546.87 μg/mL
Vehicle / solvent:
Acetone
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
EMS (Sigma; Batch BCBN1209V; Solvent DMSO)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
CP (Sigma; Batch MKBS0021V; Solvent DMSO)
Details on test system and experimental conditions:
STUDY PURPOSE
- The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line.
- The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/- was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).
- The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

CELL LINE
- The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

CELL CULTURE
- The stocks of cells are stored in liquid nitrogen at approximately -196 °C.
- Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO2 in air.
- The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Master stocks of cells were tested and found to be free of mycoplasma.

CELL CLEANSING
- The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate.
- Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

TEST ITEM PREPARATION
- Following solubility checks performed in-house, the test item was accurately weighed and
formulated in acetone prior to serial dilutions being prepared.
- The test item was considered to be a complex mixture (UVCB) and the purity of the test item was treated as 100%.
- As the test item was formulated in acetone and dosed at 0.5%, the maximum achievable dose level was set at 2500 μg/mL.
- There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings are in the table below.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test
item formulation. The test item was formulated within two hours of it being applied to the test
system. It is assumed that the formulation was stable for this duration. This was an exception with
regard to GLP and was reflected in the GLP compliance statement.

MICROSOMAL ENZYME FRACTION
- PB/BNF S9 was prepared in-house on 23 November 2014 from the livers of male Sprague Dawley rats weighing approximately 250g. These had each received, orally, three consecutive daily doses of phenobarbital/~-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process. The S9 was stored at approximately -196 °C in a liquid nitrogen freezer.
- The S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCh
(8 mM) in RO.
- S9-mix 20% (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. 10% S9-mix (i.e. 1 % final concentration of S9) was added to the cultures of Experiment 2.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E05 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with Rl 0, resuspended in R20 medium, counted and then serially diluted to 2 x 10E05 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
- The cultures were incubated at 37 °C with 5% C02 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 10E05 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

MUTAGENICITY TEST – EXPERIMENT 1
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item, 19.53 to 625 μg/mL in the absence and presence of metabolic activation, vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.1 mL of the treatment dilutions, (0.2 or 0.15 mL for the positive controls) and sufficient RO medium to bring the total volume to 20 mL.
- The treatment vessels were incubated at 3 7 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

MUTAGENICITY TEST – EXPERIMENT 2
- As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10E06 cells/mL in 10 mL cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation, the exposure period was extended to 24 hours therefore 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1 % S9 final concentration) at eight dose levels of the test item (39.06 to 625 μg/mL in the absence of metabolic activation, and 19.53 to 546.87 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 or 0.15 mL for the positive controls) and sufficient RO medium to give a final volume of 20 mL (R10 was used for the 24 hour exposure group).
- The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
- At the end of the treatment period, for each experiment, the cells were washed twice using Rl 0 medium then resuspended in R20 medium at a cell density of 2 x 10E05 cells/mL. The cultures were incubated at 37 °C with 5 % C02 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10E05 cells/mL, unless the mean cell count was less than 3 x 10E)5 cells/mL in which case all the cells were maintained.
- On Day 2 of the experiment, the cells were counted, diluted to 10E04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING
- Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation at 37 °C with 5% C02 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately Y4 to % of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
- The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth:
(i) 4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
(ii) 24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48-hour cell count/2)
(iii) Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
(iv) %RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100
CALCULATION OF DAY 2 VIABILITY (%V)
- Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method:
(i) P(0) = number of negative wells / total wells plated
(ii) %V = -ln P(0) x 100 / number of cells/well

CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
- For each culture, the relative cloning efficiency, RCE, was calculated using the equation RCE = %V / mean solvent control %V
- The RTG was then calculated for each culture using the equation RTG = (RCE * RSG) / 100

CALCULATION OF MUTATION FREQUENCY (MF)
- The mutation frequency (MF) per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
- The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

INTERPRETATION OF RESULTS
- The current historical vehicle and positive control mutation frequencies are presented in Appendix 2 (attached).
- During the course of the study dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are usually the primary factor to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values markedly less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
- For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation fre2uency value that is greater than the corresponding vehicle control by the GEF of 126 x 1 ff and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
- Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

ACCEPTABILITY OF ASSAY
- A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT are considered):
(1) The majority of the plates, for both viability (%V) and TFT resistance, are analysable for each experiment.
(2) The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
(3) The total suspension growth of the solvent control following 4-hour exposure, calculated by the day 1-fold increase in cell number multiplied by the day 2-fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range of 32 to 180.
(4) The in-house vehicle control mutant frequency is in the range of 50 to 170 x 10E-06 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10E-06 mutant frequency per survivor are not acceptable and will be repeated.
(5) Positive control chemicals (EMS and CP) should induce at least three to five-fold increases
in mutant frequency greater than the corresponding vehicle control. The positive controls should ideally yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]), of at least 300 x 10E-06 cells.
(6) The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.
(7) The highest concentration of the test item should be 10 mM or 5000 μg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurred, the highest concentration should lower the relative total growth (RTG) and/or percentage relative suspension growth (%RSG) to approximately 10 to 20 % of survival. If precipitation is noted, the highest analysed concentration should be the lowest concentration where precipitation is observed by the naked eye.
Evaluation criteria:
See above
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOTOXICITY TEST
- The dose range of the test item used in the preliminary toxicity test was 9.77 to 2500 μg/mL. The results for the Relative Suspension Growth (%RSG) were as shown in the attached table.
- There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was very sharp in all three of the exposure groups. The maximum exposure to the test item was evident around 625 μg/mL. In all three exposure groups a cloudy precipitate of the test item was observed at and above 78.13 μg/mL. A greasy oily precipitate of the test item was observed at and above 156.25 μg/mL in the 4-hour exposure groups and at and above 312.5 μg/mL in the 24-hour exposure group. The increase in %RSG was attributed to the high levels of precipitate effectively reducing exposure to the cultures. Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item-induced toxicity.

MUTAGENICITY TEST – EXPERIMENT 1
- A summary of the results from the test is presented in Table 1 (attached).
- The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7 (attached).
- There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (see Tables 3 and 6, attached).
- There was no evidence of reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred (see Tables 3 and 6, attached).
- Based on the RTG and %RSG values observed, optimum levels of toxicity were achieved in both the absence and presence of metabolic activation (see Tables 3 and 6, attached).
- In the presence of metabolic activation two dose levels 546.87 and 625 μg/mL were not plated out for TFT resistance and viability, due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances (see Tables 3 and 6, attached).
- The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see Tables 3 and 6, attached).
- The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/-locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see Tables 3 and 6, attached).
- The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10E-06 per viable cell, at any of the dose levels (see Tables 3 and 6, attached).
- It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation the test item was considered to have been adequately tested.
- A cloudy precipitate of the test item was observed at and above 156.25 which turned greasy/oily at and above 468.74 μg/mL in both the absence and presence of metabolic activation.
- The numbers of small and large colonies and their analysis are presented in Tables 4 and 7 (attached).

MUTAGENICITY TEST – EXPERIMENT 2
- The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13 (attached).
- As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 9 and 12). There was evidence of a modest reduction in viability (% V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group (Table 9). Based on the RTG and %RSG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation (see Tables 9 and 12, attached). In the absence of metabolic activation the dose levels at and above 468.74 μg/mL were not plated out for TFT resistance and viability, due to excessive toxicity. In addition 390.61 μg/mL was plated out, but later excluded from statistical analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances (see Tables 9 and 12, attached).
- The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test item.
- The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).
- The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10E-06 per viable cell, at any dose level, in either the absence or presence of metabolic activation (see Tables 9 and 12, attached). It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture.
- A cloudy precipitate of the test item was observed at and above 156.25 μg/mL which turned greasy/oily at and above 312.5 μg/mL in both exposure groups. The numbers of small and large colonies and their analysis are presented in Tables 10 and 13 (attached).
Conclusions:
The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
Executive summary:

GUIDELINE

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro” Mammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B.17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

METHODS

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, (Acetone) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies in Experiment 1 were 78.13, 156.25, 312.5, 468.74, 546.87, 625 μg/mL for 4-hour without S9 and 19.53, 39.06, 78.13, 156.25, 312.5, 468.74 μg/mL for 4-hour with S9 (2%). In Experiment 2, the dose levels plated for viability and expression of mutant colonies were 39.06, 78.13, 156.25, 312.5, 390.61 for 24-hour without S9 and 78.13, 156.25, 312.5, 390.61, 468.74, 546.87 μg/mL for 4-hour with S9 (2%).

 

RESULTS

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Overall precipitate of the test item was observed around 156.25 μg/mL in the absence and presence of metabolic activation. The vehicle controls (Acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

CONCULSION

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro

Ames test

The key study was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with an analogue test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film (creamy in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

 

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. The test item was considered to be non-mutagenic under the conditions of the test.

Chromosome aberration test

Structural chromosomal aberrations were investigated in a key study using cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 26 September 2014 and Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.

 

Duplicate cultures of human lymphocytes, treated with an analogue test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, the main experiment was performed using three exposure conditions; 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration with cell harvest after a 20-hour expression period, a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels selected for the main experiment were based on the results of the preliminary toxicity test and were limited to include the lowest precipitating dose level. The dose levels selected for the main experiment were 0,5, 10, 20, 40, 80, 160 μg/mL for the 4(20)-hour without S9, 4(20)-hour with S9 (2%) and 24-hour without S9 exposure groups.

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did demonstrate some toxicity in the preliminary toxicity test in the 24-hour exposure group only, however, the dose range for the main experiment was limited to include the lowest precipitating dose level, irrespective of toxicity, as directed in the OECD 473 guideline. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating

dose level.

 

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Mouse lymphoma assay

The study was conducted according to a method that was designed to assess the potentialmutagenicity of an analogue test item on the thymidine kinase, TK +/- locus of the L5178Y mouselymphoma cell line. The method was designed to be compatible with the OECD Guidelines forTesting of Chemicals No.476"In VitroMammalian Cell Gene Mutation Tests" adopted 21 July1997, Method B.17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPAOPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines fortesting of new chemical substances.

 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouselymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item ateight dose levels in duplicate, together with vehicle, (Acetone) and positive controls using 4-hourexposure groups both in the absence and presence of metabolic activation (2% S9). InExperiment 2, the cells were treated with the test item at eight dose levels using a 4-hourexposure group in the presence of metabolic activation(1 % S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of testitem used in the main test was selected following the results of a preliminary toxicity test. The doselevels plated for viability and expression of mutant colonies in Experiment 1 were 78.13, 156.25, 312.5,468.74, 546.87, 625 μg/mL for 4-hour without S9 and 19.53, 39.06, 78.13, 156.25, 312.5, 468.74 μg/mLfor 4-hour with S9 (2%). In Experiment 2, the dose levels plated for viability and expression of mutantcolonies were 39.06, 78.13, 156.25, 312.5, 390.61 for 24-hour without S9 and 78.13, 156.25, 312.5,390.61, 468.74, 546.87 μg/mL for 4-hour with S9 (2%).

The maximum dose levels used in the Mutagenicity Test were limited by test item-inducedtoxicity. Overall precipitate of the test item was observed around 156.25 μg/mL in the absenceand presence of metabolic activation. The vehicle controls (Acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Thepositive control treatment induced marked increases in the mutant frequency indicating thesatisfactory performance of the test and of the activityof the metabolizing system. The test item did not induce any toxicologically significant increases in themutant frequency at any of the dose levels, either with or without metabolic activation, in either thefirst or the second experiment. The test item did not induce any toxicologically significant increases in the mutant frequency atthe TK +/- locus in L5178Y cells.

In vivo

Negative results were obtained using an analogue test substance during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic and no further testing is required.

Justification for classification or non-classification

Key in vitro tests involving an analogue substance demonstrated that the test material was non-mutagenic (Ames test and mouse lymphoma assay) and non-clastogenic (chromosome aberration test). In accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic, in vivo testing is not required, and classification in accordance with Regulation (EC) No 1272/2008 does not apply.